Thin-film Phase Plates Research Articles

Thin Film Phase Plates for Cryo TEM: Fabrication, and Characterization Using Electron Holography

Thin Film Phase Plates for Cryo TEM: Fabrication, and Characterization Using Electron Holography

Oxide-free aC/Zr0.65Al0.075Cu0.275/aC phase plates for transmission electron microscopy

Thin-film phase plates (PP) have become a valuable tool for the imaging of organic objects in transmission electron microscopy (TEM). The thin film usually consists of amorphous carbon (aC), which undergoes rapid aging under intense illumination with high-energy electrons. The limited lifetime of aC film PPs calls for alternative PP materials with improved material stability. This work presents thin-film PPs fabricated from the metallic glass alloy Zr0.65Al0.075Cu0.275 (ZAC), which was identified as a promising PP material with beneficial properties, such as a large inelastic mean free path. An adverse effect of the ZAC alloy is the formation of a surface oxide layer in ambient air, which reduces the electrical conductivity and causes electrostatic charging in the electron beam. To avoid surface oxidation, the ZAC alloy is enclosed by thin aC layers. The resulting aC/ZAC/aC layer system is used to fabricate Zernike and Hilbert PPs. Phase-contrast TEM imaging is demonstrated for a sample of carbon nanotubes, which show strong contrast enhancement in PP TEM images.

Thin-Film Phase Plates for Transmission Electron Microscopy Fabricated from Metallic Glasses.

Thin-film phase plates (PPs) have become an interesting tool to enhance the contrast of weak-phase objects in transmission electron microscopy (TEM). The thin film usually consists of amorphous carbon, which suffers from quick degeneration under the intense electron-beam illumination. Recent investigations have focused on the search for alternative materials with an improved material stability. This work presents thin-film PPs fabricated from metallic glass alloys, which are characterized by a high electrical conductivity and an amorphous structure. Thin films of the zirconium-based alloy Zr65.0Al7.5Cu27.5 (ZAC) were fabricated and their phase-shifting properties were evaluated. The ZAC film was investigated by different TEM techniques, which reveal beneficial properties compared with amorphous carbon PPs. Particularly favorable is the small probability for inelastic plasmon scattering, which results from the combined effect of a moderate inelastic mean free path and a reduced film thickness due to a high mean inner potential. Small probability plasmon scattering improves contrast transfer at high spatial frequencies, which makes the ZAC alloy a promising material for PP fabrication.

The Role of Secondary Electron Emission in the Charging of Thin-Film Phase Plates

The Role of Secondary Electron Emission in the Charging of Thin-Film Phase Plates

Towards an optimum design for thin film phase plates

A variety of physical phase plate designs have been developed to maximize phase contrast for weak phase objects in the transmission electron microscope (TEM). Most progress towards application in structural biology has been made with Zernike PPs consisting of a ~30nm film of amorphous carbon with a central hole. Although problems such as beam-induced deterioration of Zernike PPs remain unsolved, it is likely that thin film phase plates will be applied routinely in TEM of ice-embedded biological specimens in the near future. However, the thick carbon film of thin film PPs dampens high-resolution information, which precludes their use for single-particle electron cryo-microscopy at atomic resolution. In this work, an improved design for a thin film phase plate is proposed, combining the advantages of Zernike PPs and 2D materials, such as graphene. The improved design features a disc of phase-shifting material mounted on an ultrathin support film. The proposed device imparts a phase shift only to electrons scattered to low angles, whereas contrast at high resolution is generated by conventional defocusing. The device maximizes phase contrast at low spatial frequencies, where defocus contrast is limiting, while damping of information at high spatial frequencies is avoided. Experiments demonstrate that the fabrication of such a device is feasible.

Towards an optimum design for electrostatic phase plates.

Charging of physical phase plates is a problem that has prevented their routine use in transmission electron microscopy of weak-phase objects. In theory, electrostatic phase plates are superior to thin-film phase plates since they do not attenuate the scattered electron beam and allow freely adjustable phase shifts. Electrostatic phase plates consist of multiple layers of conductive and insulating materials, and are thus more prone to charging than thin-film phase plates, which typically consist of only one single layer of amorphous material. We have addressed the origins of charging of Boersch phase plates and show how it can be reduced. In particular, we have performed simulations and experiments to analyze the influence of the insulating Si3N4 layers and surface charges on electrostatic charging. To optimize the performance of electrostatic phase plates, it would be desirable to fabricate electrostatic phase plates, which (i) impart a homogeneous phase shift to the unscattered electrons, (ii) have a low cut-on frequency, (iii) expose as little material to the intense unscattered beam as possible, and (iv) can be additionally polished by a focused ion-beam instrument to eliminate carbon contamination accumulated during exposure to the unscattered electron beam (Walter et al., 2012, Ultramicroscopy, 116, 62-72). We propose a new type of electrostatic phase plate that meets the above requirements and would be superior to a Boersch phase plate. It consists of three free-standing coaxial rods converging in the center of an aperture (3-fold coaxial phase plate). Simulations and preliminary experiments with modified Boersch phase plates indicate that the fabrication of a 3-fold coaxial phase plate is feasible.

Charging of Thin Film Phase Plates under Electron Beam Irradiation

Charging of Thin Film Phase Plates under Electron Beam Irradiation

True Causes of Charging and Anti-Charging Conditions in Thin-Film Phase Plates

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Practical aspects of Boersch phase contrast electron microscopy of biological specimens

Implementation of physical phase plates into transmission electron microscopes to achieve in-focus contrast for ice-embedded biological specimens poses several technological challenges. During the last decade several phase plates designs have been introduced and tested for electron cryo-microscopy (cryoEM), including thin film (Zernike) phase plates and electrostatic devices. Boersch phase plates (BPPs) are electrostatic einzel lenses shifting the phase of the unscattered beam by an arbitrary angle. Adjusting the phase shift to 90° achieves the maximum contrast transfer for phase objects such as biomolecules. Recently, we reported the implementation of a BPP into a dedicated phase contrast aberration-corrected electron microscope (PACEM) and demonstrated its use to generate in-focus contrast of frozen-hydrated specimens. However, a number of obstacles need to be overcome before BPPs can be used routinely, mostly related to the phase plate devices themselves. CryoEM with a physical phase plate is affected by electrostatic charging, obliteration of low spatial frequencies, and mechanical drift. Furthermore, BPPs introduce single sideband contrast (SSB), due to the obstruction of Friedel mates in the diffraction pattern. In this study we address the technical obstacles in detail and show how they may be overcome. We use X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) to identify contaminants responsible for electrostatic charging, which occurs with most phase plates. We demonstrate that obstruction of low-resolution features is significantly reduced by lowering the acceleration voltage of the microscope. Finally, we present computational approaches to correct BPP images for SSB contrast and to compensate for mechanical drift of the BPP.

Retrofit implementation of Zernike phase plate imaging for cryo-TEM

In-focus phase-plate imaging is particularly beneficial for cryo-TEM because it offers a substantial overall increase in image contrast, without an electron dose penalty, and it simplifies image interpretation. We show how phase-plate cryo-TEM can be implemented with an appropriate existing TEM, and provide a basic practical introduction to use of thin-film (carbon) phase plates. We point out potential pitfalls of phase-plate operation, and discuss solutions. We provide information on evaluating a particular TEM for its suitability.

Phase Contrast Enhancement with Phase Plates in Biological Electron Microscopy

Theoretically, transmission electron microscopy (TEM) is compatible with three different types of phase plate: thin-film, electrostatic, and magnetic. However, designing functional phase plates has been an arduous process that has suffered from unavoidable technical obstacles such as phase-plate charging and difficulties associated with micro-fabrication of electrostatic and magnetic phase plates. This review discusses phase-contrast schemes that allow visualization of transparent objects with high contrast. Next it deals with recent studies on biological applications ranging from proteins and viruses to whole cells. Finally, future prospects for overcoming the problem of phase-plate charging and for designing the next generation of phase-plates to solve the problem of electron loss inherent in thin-film phase plates are discussed.

Phase-plate electron microscopy: a novel imaging tool to reveal close-to-life nano-structures

After slow progress in the efforts to develop phase plates for electron microscopes, functional phase plate using thin carbon film has been reported recently. It permits collecting high-contrast images of close-to-life biological structures with cryo-fixation and without staining. This report reviews the state of the art for phase plates and what is innovated with them in biological electron microscopy. The extension of thin-film phase plates to the material-less type using electrostatic field or magnetic field is also addressed.Electronic supplementary materialThe online version of this article (doi:10.1007/s12551-008-0006-z) contains supplementary material, which is available to authorized users.

Phase contrast electron microscopy: development of thin-film phase plates and biological applications

Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is focused on two techniques that employ two different types of phase plates. The first technique uses a Zernike phase plate, which is made of a uniform amorphous carbon film that completely covers the aperture of an objective lens and can retard the phase of electron waves by π/2, except at the centre where a tiny hole is drilled. The other technique uses a Hilbert phase plate, which is made of an amorphous carbon film that is twice as thick as the Zernike phase plate, covers only half of the aperture and retards the electron wave phase by π. By combining the power of efficient phase contrast detection with the accurate preservation achieved by a cryotechnique such as vitrification, macromolecular complexes and supermolecular structures inside intact bacterial or eukaryotic cells may be visualized without staining. Phase contrast cryo-TEM has the potential to bridge the gap between cellular and molecular biology in terms of high-resolution visualization. Examples using proteins, viruses, cyanobacteria and somatic cells are provided.

Development of phase plates for electron microscopes and their biological application

After slow progress in the efforts to develop phase plates for electron microscopes, functional phase plates with thin carbon films have recently been reported. An electron microscope enhanced with thin-film phase plates has practical advantages. It permits collecting high-contrast images of intact biological specimens without harsh and lengthy sample preparation, such as fixation, dehydration, resin-embedding, staining and thin-sectioning. This report reviews the state of the art for phase plates in biological electron microscopy and focuses upon the conditions required for functional thin-film phase plates. The current disadvantages of thin-film phase plates are also addressed and potential solutions are proposed.